Primary human gingival fibroblasts (HGF-1) were previously established in the lab (IRB # 911778-10) and purchased from ATCC (CRL-2014). Cells were cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum and 100 UI/mL penicillin/streptomycin. HGF were cultured in a 5% CO2 incubator at 37°C. The growth media was replaced every 2-3 days. Experiments were performed using cells between the 4th and 8th passages, at approximately 80% confluence detailed in figure legends. The fibroblast phenotype was confirmed by positive staining for vimentin and FSP1. Viable cells were automatically counted using a cell counter and Trypan Blue staining and seeded in uniformity of cell distribution in OptiMEM medium the day before each experiment. For cell treatments, the following reagents were used: 1ng/mL human recombinant IL-1β (201LB005, R&D systems); 100μM Adenosine (A4036-5G, MilliporeSigma); 30μM EHNA (E114-25MG, MilliporeSigma), 1μM FCCP (S8276, Selleck Chemicals LLC) and 10μM SR-18292 (HY-101491, MedChem Express). Pre-treatment with drugs was performed 1 hour prior to the adenosine or EHNA+adenosine stimulation. EHNA was added 5min prior to adenosine. Adenosine was added 5 min prior to IL-1β and cells were incubated for the time points indicated in each figure legend.
Sr 18292
Manufactured by MedChemExpress
Sourced in United States, China
SR-18292 is a laboratory reagent used for research purposes. It functions as a selective cannabinoid CB1 receptor antagonist. Further details on its core function or intended use are not available.
Automatically generated - may contain errors
Lab products found in correlation
Ex527, by MedChemExpress (2 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Gentamicin, by MedChemExpress (1 mentions)
Gentamicin, by Meilun (1 mentions)
Mouse endothelial culture medium, by Cell Biologics (1 mentions)
Dynabead, by Thermo Fisher Scientific (1 mentions)
Anti cd31 antibody, by Thermo Fisher Scientific (1 mentions)
Collagenase 1, by Worthington (1 mentions)
Mitochondrial membrane potential assay kit with jc 1, by Beyotime (1 mentions)
Anti acetylated lysine, by Cell Signaling Technology (1 mentions)
Enhanced atp assay kit, by Beyotime (1 mentions)
Tissue mitochondria isolation kit, by Beyotime (1 mentions)
Srt1720, by MedChemExpress (1 mentions)
Tl02 59, by MedChemExpress (1 mentions)
Lipopolysaccharide lps, by Merck Group (1 mentions)
Zln005, by MedChemExpress (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
β actin, by Bioworld Technology (1 mentions)
Lipofectamine 6000, by Beyotime (1 mentions)
10 protocols using sr 18292
1
Gingival Fibroblast Adenosine Signaling
2
UA Protects NPMSCs from H2O2-Induced Damage
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NPMSCs were seeded in 96-well plates at a density of 2 × 103 cells per well and incubated in complete medium overnight at 37 °C with 5% CO2. Then, NPMSCs were treated with different concentrations ((0-80 μM, 0-48 h) of UA (MedChem Express, China, catalog No. 1143-70-0). After that, 10% cell counting kit-8 (CCK-8) (Dojindo, Japan, catalog No. CK04) was added to each well at different time points, and the optical density (OD) value was read after 1 h of incubation at 480 nm by a microplate reader (Bio-Rad, United States). Cell viability was calculated as follows: Cell viability (of control) = [(Ae-Ab)/(Ac-Ab)]. Ae, Ab, and Ac represent the OD value of the treatment, blank and control groups, respectively. Similarly, to further determine whether UA works through the silent information regulator of transcription 1/PPAR gamma coactivator-1α (SIRT1/PGC-1α) pathway, different concentrations (0-160 μM, 0-36 h) of the PGC-1α inhibitor SR-18292 (MedChem Express, China, catalog No. HY-101491) were cocultured with NPMSCs.
NPMSCs were divided into four groups in the following examinations: (1) Control group; (2) H2O2 group (80 μM H2O2); (3) H2O2 + UA group (80 μM H2O2 + 20 μM UA); and (4) H2O2 + UA + SR-18292 group (80 μM H2O2 + 20 μM UA + 20 μM SR-18292).
NPMSCs were divided into four groups in the following examinations: (1) Control group; (2) H2O2 group (80 μM H2O2); (3) H2O2 + UA group (80 μM H2O2 + 20 μM UA); and (4) H2O2 + UA + SR-18292 group (80 μM H2O2 + 20 μM UA + 20 μM SR-18292).
3
Gentamicin-Induced Ototoxicity Model
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Ototoxicity models were established in HEI-OC1 cells and cochlear explants by treatment for 12 h with 500 μM gentamicin (Meilun Biotechnology, Dalian, China). To evaluate the effects of DHM, samples were pretreated with 1 mM DHM (Meilun Biotechnology) for 24 h before adding gentamicin. To evaluate the role(s) of PGC-1α and SIRT3 in the protective effect of DHM against gentamicin-induced ototoxicity, samples were pretreated with the PGC-1α and SIRT3 inhibitors SR-18292 (20 μM; MedChemExpress, Princeton, NJ, United States) and 3-(1H-1,2,3-triazol-4-yl) pyridine (3-TYP, 50 μM; Absin Biotechnology), respectively, for 2 h before the addition of DHM and exposure to gentamicin.
4
Isolation and Culture of Mouse Cardiac Endothelial Cells
5
Investigating AMPK and PGC1α Inhibitors in Muscle Cell Differentiation
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Dorsomorphin dihydrochloride (Compound C dihydrochloride, 10 μM, MedChemExpress, China) [27 (link)] was used to inhibit the activity of AMPK and SR-18292 (20 μM, MedChemExpress, China) [28 (link)] was used to inhibit the activity of PGC1α, respectively. After 5 days of differentiation, C2C12 cells were incubated with the intervention for 18 h in the CON group (PBS), DEX group (100 μM dexamethasone), DG group (100 μM dexamethasone + 5 × 108 particles/mL GqDNVs), CC group (10 μM Compound C dihydrochloride + 100 μM dexamethasone + 5 × 108 particles/mL GqDNVs) and SR group (20 μM SR-18292 + 100 μM dexamethasone + 5 × 108 particles/mL GqDNVs).
6
PGC-1α Inhibitor Effects on Mitochondrial Function
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The PGC-1a inhibitor SR-18292 (#YH-101491) was obtained from MedChemExpress (NJ, USA). Nissl staining solution (Cresyl Violet) (#G1430) and mitochondrial respiration complex I (#BC0515) and II (#BC3230) activity assay kits were purchased from Beijing Solarbio Science & Technology (Beijing, China), and the in-situ cell detection kit (AP, #11684809910) for terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) staining was purchased from Roche Diagnostics (Minn, USA). Tissue mitochondria isolation kit (#C3606), mitochondrial membrane potential assay kit with JC-1 (#C2006), and enhanced ATP assay kit (#S0027) were provided by Beyotime Institute of Biotechnology (Nanjing, China). Antibodies against PGC-1α, NRF2, and Tfam were obtained from Abcam (Cambridge, UK), anti-acetylated-lysine was provided by Cell Signalling Technology (Mass, USA), the antibody against β-actin was purchased from Hangzhou HuaAn Biotechnology (Hangzhou, China), and goat antimouse IgG and goat anti-rabbit IgG antibodies were from KPL (Md, USA).
7
Pharmacological Modulation of Sirtuin and PGC-1α Pathways
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TL02-59 (an Fgr inhibitor), (R) EX-527 (a SIRT1 inhibitor), SRT 1720 (a SIRT1 activator), ZLN005 (a PGC-1α activator), and SR-18292 (a PGC-1α inhibitor) were obtained from MedChemExpress (Monmouth Junction, NJ, USA). Lipopolysaccharide (LPS) was purchased from Sigma (St. Louis, MO, USA).
TL02-59 was dissolved in 90% saline, 5% solution HS-15, and 5% N-methyl-2-pyrrolidone. TL02-59 (at 1 mg/kg, 10 mg/kg, or 15 mg/kg) and vehicle were administrated once daily for 3 days via the tail vein at 1 h after CLP. The CLP mice were injected i.p. with ZLN005 or SR-18292 at 12 mg/kg or 45 mg/kg after TL02-59 treatment. In the HT22 cells, TL02-59 (1 μM) was administered to cells for 24 h in the presence or absence of LPS (1 μg/mL). To determine whether (R) EX-527 or SRT 1720 treatment could reverse the protective effects of TL02-59, HT22 cells were incubated with (R) EX-527 (10 μM) or SRT 1720 (1 μM) for 24 h in the presence of LPS and TL02-59. To determine whether ZLN005 or SR 18292 treatment could reverse the protective effects of TL02-59, HT22 cells were incubated with ZLN005 (2 μM) or SR 18292 (2.5 μM) for 24 h in the presence of LPS and TL02-59.
TL02-59 was dissolved in 90% saline, 5% solution HS-15, and 5% N-methyl-2-pyrrolidone. TL02-59 (at 1 mg/kg, 10 mg/kg, or 15 mg/kg) and vehicle were administrated once daily for 3 days via the tail vein at 1 h after CLP. The CLP mice were injected i.p. with ZLN005 or SR-18292 at 12 mg/kg or 45 mg/kg after TL02-59 treatment. In the HT22 cells, TL02-59 (1 μM) was administered to cells for 24 h in the presence or absence of LPS (1 μg/mL). To determine whether (R) EX-527 or SRT 1720 treatment could reverse the protective effects of TL02-59, HT22 cells were incubated with (R) EX-527 (10 μM) or SRT 1720 (1 μM) for 24 h in the presence of LPS and TL02-59. To determine whether ZLN005 or SR 18292 treatment could reverse the protective effects of TL02-59, HT22 cells were incubated with ZLN005 (2 μM) or SR 18292 (2.5 μM) for 24 h in the presence of LPS and TL02-59.
8
Glucose and Lipid Metabolism in MIN6 Cells
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Mouse pancreatic β-cell line MIN6 (Keygen Biotech, Nanjing, China) cells were cultured in RPMI1640 medium (Gibco) accompanied with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin. Cells were passaged every three days. Culture media was changed every 24 h.
For intracellular signaling studies, MIN6 cells were starved for 12 h followed by incubation with or without ∆nFGF1 (50 ng/mL) for 1 h. Cells were then exposed to media containing normal glucose (NG, 11.1 mM) as a control or high glucose (HG, 33 mM)+palmitic acid (PA, 0.5 mM) for 24 h and then lysed to detect protein expression by Western blot. For inhibitor experiments, MIN6 cells were also starved for 12 h and treated with AMPK inhibitor Compound C (10 μM, Selleck Chemicals, S7306) or SIRT1 inhibitor EX-527 (10 μM, MedChemExpress, HY-15452) or PGC-1α inhibitor SR-18292 (10 μM, MedChemExpress, HY-101491) for 1 h and then incubated in NG or HG+PA or HG+PA+∆nFGF1 for 24 h and lysed to detect protein expression by Western blot. For siRNA knockdown experiments, MIN6 cells were seeded and grown in six-well plates for 24 h to achieve 70% confluence. Cell transfection was performed with the transfection reagent Lipofectamine 3000 in accordance with the manufacturer's instructions. A 24 h transfection of AMPK siRNA (Santa Cruz Biotechnology, sc45313) was followed by starvation and treatment as described above.
For intracellular signaling studies, MIN6 cells were starved for 12 h followed by incubation with or without ∆nFGF1 (50 ng/mL) for 1 h. Cells were then exposed to media containing normal glucose (NG, 11.1 mM) as a control or high glucose (HG, 33 mM)+palmitic acid (PA, 0.5 mM) for 24 h and then lysed to detect protein expression by Western blot. For inhibitor experiments, MIN6 cells were also starved for 12 h and treated with AMPK inhibitor Compound C (10 μM, Selleck Chemicals, S7306) or SIRT1 inhibitor EX-527 (10 μM, MedChemExpress, HY-15452) or PGC-1α inhibitor SR-18292 (10 μM, MedChemExpress, HY-101491) for 1 h and then incubated in NG or HG+PA or HG+PA+∆nFGF1 for 24 h and lysed to detect protein expression by Western blot. For siRNA knockdown experiments, MIN6 cells were seeded and grown in six-well plates for 24 h to achieve 70% confluence. Cell transfection was performed with the transfection reagent Lipofectamine 3000 in accordance with the manufacturer's instructions. A 24 h transfection of AMPK siRNA (Santa Cruz Biotechnology, sc45313) was followed by starvation and treatment as described above.
9
Mitochondrial Fusion Modulator Protocol
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Mitochondrial fusion promoter M1, which promotes mitochondrial fusion by increasing the expression of ATP synthase 5A/B [16 (link)], was purchased from Sigma-Aldrich (St. Louis, USA). SB-431542, (E)-SIS3 and SR-18292 were purchased from MedChemExpress (Houston, USA), CD4+CD62L+T Cell Isolation Kit was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Lipofectamine 6000 was purchased from Beyotime Biotechnology (Shanghai, China). FITC-anti-CD4, APC-antiCD25, PE-anti-Foxp3, and purified anti-mouse CD3/CD28 mAbs were purchased from eBioscience (San Diego, USA). Transforming growth factor-β1 (TGF-β1) was purchased from R&D Systems (Minneapolis, USA). Murine IL-2 was purchased from PeproTech (Madison, USA). Primary antibodies against Mfn1, Mfn2, Opa1 and CPT1 were purchased from Santa Cruz Biotechnology (California, USA). Primary antibody against HK2 was purchased from Affinity Biosciences (OH, USA). Primary antibodies against Smad2, Smad3, p-Smad2, p-Smad3, PGC-1α and β-actin were purchased from Bioworld Technology (Georgia, USA). Freund's complete adjuvant was purchased from Sigma-Aldrich (St. Louis, USA). Dextran sodium sulfate (DSS, MW 36–50 kDa) was purchased from MP Biomedical (Aurora, USA). Myeloperoxidase (MPO) kit was purchased from Jiancheng Biotech (Nanjing, China). TRIzol reagent was purchased from SunShine Biotechnology (Nanjing, China).
10
PGC1α Inhibitor and SIRT3 Overexpression in LPS-Treated Cells
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At about 50% confluence, cells were first incubated with PGC1α inhibitor SR-18292 (100 μM, HY-101491, MedChemExpress) for 6 h, then with or without 50 MOI Ad-SIRT3 infection for 6 h, followed by treatment with 150 μg/mL LPS for 12 h (n = 3). Cells were harvested by trypsin digestion and stored for further analysis.
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